Excretory overexpression of hydrophobins as multifunctional biosurfactants in E. coli

Cui, Lupeng; Cheng, Cheng; Qiu, Yumeng; Jiang, Tianyue; He, Bingfang

doi:10.1016/j.ijbiomac.2020.09.206

Cited by 3 publications

(1 citation statement)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Owing to small size, the hydrophobin tagged proteins are secreted to the culture for rapid puri cation from different expression hosts including the green microalga and yeast [22,23]. I is fused with the partner Ffu312 is produced in E. coli, and excreted into the culture medium for simple puri cation [24]. The inverse temperature transition for yeast secreted production of the sole ELP is pH dependent [25].…”

Section: Introductionmentioning

confidence: 99%

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Chen,

Li,

et al. 2024

Preprint

View full text Add to dashboard Cite

Background Rapid and cost-effective purification of the target protein for extracellular production in Escherichia coli is still challenge. Previously, we identified that human annexin A1 as a N-terminal fusion tag for Ca2+-dependent phase transition to simply, rapidly and cost-effectively purify three fluorescent proteins including emerald green fluorescent protein (EmGFP), red fluorescent protein mCherry, and flavin-binding cyan-green fluorescent protein.Results When the phage lytic protein was induced later, the annexin A1 tagged EmGFP was leaked into the culture, but purification efficiency was relatively low. Pre-overexpression of Bacillus cereus phospholipase C facilitated intracellular production of the fusion protein, and purified fusion protein showed the purity higher than other two fluorescent protein fusions. Using the co-expression system, the elastin-like polypeptide (ELP) tagged three fluorescent proteins via extracellular production were also purified in revisable protein precipitation. The yield of the purified annexin A1 tagged protein was comparable to that of the purified ELP tagged one. The silica-binding peptide tagged annexin A1-EmGFP bound to silica particles, and the ELP tagged mCherry strongly bound to phenyl sepharose was efficient for column-dependent purification. The extracellular nine tobacco etch virus protease variants with the annexin A1 tag were purified and the cleavage activity was assayed. Using the purified protease variant with the highest activity, the purification tag was removed in solution, or by on-resin cleavage of the immobilized annexin A1 or ELP tagged EmGFP. The soluble annexin A1-EmGFP with the Bacillus amyloliquefaciens alpha-amylase signal peptide was poorly produced in Bacillus subtilis, and the fusion protein with the α-factor signal peptide was located in intracellular Pichia pastoris.Conclusions The annexin A1 or ELP fusions in the culture were purified by revise transition cycles. On-resin cleavage facilitated removal of the reagents for protein purification, and fusion tag. However, the annexin A1-EmGFP fused the correspondent signal peptides displayed poor secretion efficiency in B. subtilis and P. pastoris. The platform will be used for simply and cost-effectively purifying the target proteins with industrial and clinical values without cell disruption process, and rapidly testifying the activity of the engineered enzyme variants.

show abstract

Section: Introductionmentioning

confidence: 99%

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Chen,

Li,

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

Excretory expression of IsPETase in E. coli by an enhancer of signal peptides and enhanced PET hydrolysis

Cui

Qiu

Liang

et al. 2021

International Journal of Biological Macromolecules

View full text Add to dashboard Cite

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy

Tian,

Feng,

Wei

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

show abstract

Excretory overexpression of hydrophobins as multifunctional biosurfactants in E. coli

Cited by 3 publications

References 39 publications

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Excretory expression of IsPETase in E. coli by an enhancer of signal peptides and enhanced PET hydrolysis

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy

Contact Info

Product

Resources

About